The LH, FSH and TSH receptors, collectively known as the glycoprotein hormone receptor (GPH-R's), comprise a unique subfamily of rhodopsin-like G protein-coupled receptors (GPCR's). They are each composed of a large extracellular domain that binds hormone and a seven transmembrane (TM) domain that couples to G proteins, primarily Gs. The active states of each of these receptors are stabilized by the binding of hormone or by discrete mutations that induce constitutive activity. The aims of this grant are to determine the structural basis for the activation of the GPH-R's. In general, we will use two experimental approaches, each of which will be coupled with molecular modeling. The first approach utilizes disruptive and reciprocal mutagenesis to test the hypothesis that TM residues that are highly conserved in the three GPH-R's participate in interhelical interactions that stabilize the receptors in the resting state. The second approach uses selective chimera mutagenesis between different GPH-R's from a given species or from a given GPH-R of different species to test the hypothesis that certain divergent TM residues modulate the activities of the GPH-R's by affecting interhelical interactions. In spite of the high degree of amino acid identity in the TM regions of the GPH-R's, certain pairs of GPH-R's exhibit markedly different degrees of basal activity, mutation-induced, or hormone-induced activation. By interchanging the divergent TM residues between two related GPH-R's with differing properties, we can maintain the overall integrity of the receptor (because the divergent residues are capable of maintaining the overall structure) and determine the precise amino acids that confer the greater or lesser degree of activity. Computer modeling will be coupled with each of these experimental strategies to aid in data interpretation and to define those interhelical interactions that stabilize the GPH-R's in the resting states, in mutation-induced active states, and in hormone-stabilized active states. The specific aims of this proposal are: 1. Determine the roles that conserved residues of the GPH-R's have in maintaining the resting states of the receptors. 2. Determine the structural basis for the different degrees of constitutive activity of the resting states of the GPH-R's. 3. Determine the structural basis for the different susceptibilities of the GPH-R's to be stabilized in an active state by activating mutations. 4. Determine the structural basis for the different degrees of hormone-stimulated Gs activation by GPH-R's.